Glaucoma is a major cause of blindness. Elevated intraocular pressure (IOP) is the primary risk factor for glaucomatous optic nerve damage and reducing IOP remains the only treatment for all forms of glaucoma. Loss of the ability to appropriately regulate the outflow resistance, i.e. to maintain IOP homeostasis, due to a variety of genetic or environmental causes, is a hallmark of much of glaucoma. Understanding the mechanisms regulating IOP homeostasis, which is the focus of this proposal, is thus central to improved function based therapy for this common blinding disease. Our prior studies point to the following framework for IOP regulation. Extracellular matrix (ECM) turnover, initiated by trabecular meshwork (TM) matrix metalloproteinases (MMPs) is required to maintain the aqueous humor outflow resistance and thus IOP. In response to a significant and sustained pressure change, the outflow pathway initiates an IOP homeostatic response in which adjustments are made to the outflow resistance, thus restoring IOP to within a narrow normal range. TM ECM turnover is central to this process. Sustained pressure changes are sensed by cells within the juxtacanalicular region of the TM and/or Schlemm's canal (SC) inner wall as mechanical stretching or distortion. These cells then initiated a complex program of ECM turnover to adjust the outflow resistance over several days' time and restore IOP to within acceptable bounds. In addition, outflow is highly segmental around the circumference of the eye, which has dramatic consequences for understanding all aspects of outflow facility and the mechanisms of IOP homeostasis. We propose two aims focused on unraveling the molecular mechanisms responsible for regulating IOP homeostatic outflow resistance adjustments. These studies will rely primarily on TM and SC cell culture and perfused anterior segment organ culture. Specific Aim 1 will entail detailed molecular comparisons of high flow regions with low flow regions for 1x vs. 2x perfusion pressures at a series of time points during which the IOP homeostatic resistance adjustment is occurring using both normal and glaucoma eyes. Methods will include: quantitative RT-PCR, PCR arrays, direct regional dissection, and laser capture microdissection as well as confocal immunohistochemistry, Western immunoblots, and ELISAs. Specific Aim 2 will be to identify the signal transduction pathways that regulate the regional outflow resistance changes that occur in response to the 1x to 2x pressure change. This will include assessing activation states of key components of select signal transduction pathways and transcriptional modulators. Verification of involvement in the IOP homeostatic process will include using pathway inhibitors or activators and genetic manipulation of pathway components via RNAi and gene overexpression to modulate specific pathway components and thus affect the IOP homeostatic process. This detailed molecular and cellular understanding of how the IOP homeostatic process is regulated in normal and glaucomatous eyes will provide new targets to restore this homeostatic process in glaucomatous eyes.